The focus of this project is Francisella tularensis, the causative agent of tularemia and a Category A Bioterrorism Select Agent. There is an urgent need for data and reagents that immediately can be directed toward vaccine and therapeutic agent discovery as well as immunological diagnostic assay development for F. tularensis, and other Category A organisms. Our overall goal is to use genomic and proteomic approaches to identify genes involved in F. tularensis pathogenesis and antigens important for tularemia immunity. In this proposal, we will merge novel sequencing and oligosynthesis/array technologies (developed by our subcontract partners) with traditional Sanger sequencing strategies to create a cost effective method to identify genetic differences between important Francisella strains with different virulence and lifestyle phenotypes. As a first step toward this goal, we will determine the genomic sequences of two type A and one type B Francisella strain. These sequences will be compared to three already completed Francisella genome sequences to identify the insertions, deletions, and single-nucleotide polymorphisms that may lead to differing Francisella phenotypes. Genome sequencing will be coupled to proteome expression using a novel shotgun library/expression plasmid to both host the clones for Francisella sequencing and to express peptides from each Francisella proteome. Peptides from the three proteomes will be screened with immunoreactive sera to identify differences in their antigen profiles. The comparative genetic analysis of virulent and avirulent Francisella strains in conjunction with an antigen screen against peptides from the proteomes of these bacteria will identify putative virulence factors and antigens for future study as vaccine and immunodiagnostic reagents.